Pharmaceutically active isoindoline derivatives

ABSTRACT

Isoindolin-1-one and Isoindoline-1,3-dione substituted in the 2-position with a 2,6-dioxo-3-hydroxypiperidin-5-yl group, which may be further substituted in the 5-position with alkyl or halogeno, and in the 4-position with alkyl or a nitrogen-containing group are inhibitors of, and thus useful in the treatment of disease states mediated by, TNFα. A typical embodiment is 2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-aminoisoindolin-1-one.

This application is a continuation of application Ser. No. 10/262,210,filed Sep. 30, 2002 now U.S. Pat. No. 6,762,195, which is a Continuationof Ser. No. 09/712,550, filed Nov. 14, 2000, now U.S. Pat. No.6,458,810, the disclosure of each of which is incorporated by referenceherein in its entirety.

The present invention pertains to non-polypeptide isoindolinederivatives that decrease the levels of tumor necrosis factor alpha(TNFα) and to the treatment of disease states mediated thereby. Thecompounds inhibit angiogenesis and are useful in the treatment ofcancer, inflammatory, and autoimmune diseases. For example, compoundsthat selectively inhibit TNFα are useful in treating inflammation andeffecting relaxation of airway smooth muscle with a minimum of unwantedside effects, e.g., cardiovascular or anti-platelet effects. The presentinvention also relates to methods of treatment and pharmaceuticalcompositions utilizing such compounds.

BACKGROUND OF THE INVENTION

Tumor necrosis factor α, or TNFα, is a cytokine which is releasedprimarily by mononuclear phagocytes in response to a numberimmunostimulators. When administered to animals or humans, it causesinflammation, fever, cardiovascular effects, hemorrhage, coagulation,and acute phase responses similar to those seen during acute infectionsand shock states. Excessive or unregulated TNFα production thus has beenimplicated in a number of disease conditions. These include endotoxemiaand/or toxic shock syndrome {Tracey et al., Nature 330, 662–664 (1987)and Hinshaw et al., Circ. Shock 30, 279–292 (1990)}; rheumatoidarthritis, Crohn's disease, IBD, cachexia {Dezube et al., Lancet, 335(8690), 662 (1990)} and Adult Respiratory Distress Syndrome where TNFαconcentration in excess of 12,000 pg/mL have been detected in pulmonaryaspirates from ARDS patients {Millar et al., Lancet 2(8665), 712–714(1989)}. Systemic infusion of recombinant TNFα also resulted in changestypically seen in ARDS {Ferrai-Baliviera et al., Arch. Surg. 124(12),1400–1405 (1989)}.

TNFα appears to be involved in bone resorption diseases, includingarthritis. When activated, leukocytes will produce bone-resorption, anactivity to which the data suggest TNFα contributes. {Bertolini et al.,Nature 319, 516–518 (1986) and Johnson et al., Endocrinology 124(3),1424–1427 (1989)}. TNFα also has been shown to stimulate bone resorptionand inhibit bone formation in vitro and in vivo through stimulation ofosteoblast formation and activation combined with inhibition ofosteoblast function. Although TNFα may be involved in many boneresorption diseases, including arthritis, a most compelling link withdisease is the association between production of TNFα by tumor or hosttissues and malignancy associated hypercalcemia {Calci. Tissue Int. (US)46(Suppl.), S3-10 (1990)}. In Graft versus Host Reaction, increasedserum TNFα levels have been associated with major complication followingacute allogenic bone marrow transplants {Holler et al., Blood, 75(4),1011–1016 (1990)}.

Cerebral malaria is a lethal hyperacute neurological syndrome associatedwith high blood levels of TNFα and the most severe complicationoccurring in malaria patients. Levels of serum TNFα correlated directlywith the severity of disease and the prognosis in patients with acutemalaria attacks {Grau et al., N. Engl. J. Med. 320(24), 1586–1591(1989)}.

Unregulated angiogenesis is pathologic and sustains progression of manyneo-plastic and non-neoplastic diseases including solid tumor growth andmetastases, arthritis, some types of eye disorders, and psoriasis. See,e.g., Moses et al., 1991, Biotech. 9:630–634; Folkman et al., 1995, N.Engl. J. Med., 333:1757–1763; Auerbach et al., 1985, J. Microvasc. Res.29:401–411; Folkman, 1985, Advances in Cancer Research, eds. Klein andWeinhouse, Academic Press, New York, pp. 175–203; Patz, 1982, Am. J.Opthalmol. 94:715–743; Folkman et al., 1983, Science 221:719–725; andFolkman and Klagsbrun, 1987, Science 235:442–447. In addition,maintenance of the avascularity of the cornea, lens, and trabecularmeshwork is crucial for vision as well as to ocular physiology. See,e.g., reviews by Waltman et al., 1978, Am. J. Ophthal. 85:704–710 andGartner et al., 1978, Surv. Ophthal. 22:291–312.

Angiogenesis thus is encountered in various disease states, tumormetastasis, and abnormal growth by endothelial cells. Pathologicalstates created by unregulated angiogenesis have been grouped together asangiogenic dependent or angiogenic associated diseases. Control of theangiogenic processes could lead to the mitigation of these conditions.

The components of angiogenesis relating to vascular endothelial cellproliferation, migration and invasion, have been found to be regulatedin part by polypeptide growth factors. Endothelial cells exposed to amedium containing suitable growth factors can be induced to evoke someor all of the angiogenic responses. Polypeptides with in vitroendothelial growth promoting activity include acidic and basicfibroblast growth factors, transforming growth factors α and β,platelet-derived endothelial cell growth factor, granulocytecolony-stimulating factor, interleukin-8, hepatocyte growth factor,proliferin, vascular endothelial growth factor and placental growthfactor. Folkman et al, 1995, N. Engl. J. Med., 333:1757–1763.

Inhibitory influences predominate in the naturally occurring balancebetween endogenous stimulators and inhibitors of angiogenesis.Rastinejad et al., 1989, Cell 56:345–355. In those instances in whichneovascularization occurs under normal physiological conditions, such aswound healing, organ regeneration, embryonic development, and femalereproductive processes, angiogenesis is stringently regulated andspatially and temporally delimited. Under conditions of pathologicalangiogenesis such as that characterizing solid tumor growth, theseregulatory controls fail.

Macrophage-induced angiogenesis is known to be mediated by TNFα.Leibovich et al. {Nature, 329, 630–632 (1987)} showed TNFα induces invivo capillary blood vessel formation in the rat cornea and thedeveloping chick chorioallantoic membranes at very low doses and suggestTNFα is a candidate for inducing angiogenesis in inflammation, woundrepair, and tumor growth.

TNFα production also has been independently associated with cancerousconditions, particularly induced tumors {Ching et al., Brit. J. Cancer,(1955) 72, 339–343, and Koch, Progress in Medicinal Chemistry, 22,166–242 (1985)}. Whether or not involved with TNFα production,angiogenesis is prominent in solid tumor formation and metastasis andangiogenic factors have been found associated with several solid tumorssuch as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma, neuroblastoma,and osteosarcoma. Tumors in which angiogenesis is important includesolid tumors, and benign tumors such as acoustic neuroma, neurofibroma,trachoma and pyogenic granulomas. Independent of its action on TNFαproduction, the prevention of angiogenesis could halt the growth ofthese tumors and the resultant damage to the animal due to the presenceof the tumor. Angiogenesis has been associated with blood-born tumorssuch as leukemias and various acute or chronic neoplastic diseases ofthe bone marrow. In such conditions, unrestrained proliferation of whiteblood cells occurs, usually accompanied by anemia, impaired bloodclotting, and enlargement of the lymph nodes, liver, and spleen.

Angiogenesis also is involved in tumor metastasis. Thus angiogenesisstimulation occurs in vascularization of the tumor, allowing tumor cellsto enter the blood stream and circulate throughout the body. After thetumor cells have left the primary site, and have settled into thesecondary, metastasis site, angiogenesis must occur before the new tumorcan grow and expand.

All of the various cell types of the body can be transformed into benignor malignant tumor cells. The most frequent tumor site is lung, followedby colorectal, breast, prostate, bladder, pancreas, and then ovary.Other prevalent types of cancer include leukemia, central nervous systemcancers, including brain cancer, melanoma, lymphoma, erythroleukemia,uterine cancer, and head and neck cancer.

TNFα also plays a role in the area of chronic pulmonary inflammatorydiseases. The deposition of silica particles leads to silicosis, adisease of progressive respiratory failure caused by a fibroticreaction. Antibody to TNFα completely blocked the silica-induced lungfibrosis in mice {Pignet et al., Nature, 344:245–247 (1990)}. Highlevels of TNFα production (in the serum and in isolated macrophages)have been demonstrated in animal models of silica and asbestos inducedfibrosis {Bissonnette et al., Inflammation 13(3), 329–339 (1989)}.Alveolar macrophages from pulmonary sarcoidosis patients have also beenfound to spontaneously release massive quantities of TNFα as comparedwith macrophages from normal donors {Baughman et al., J. Lab. Clin. Med.115(I), 36–42 (1990)}.

TNFα is also implicated in the inflammatory response which followsreperfusion, called reperfusion injury, and is a major cause of tissuedamage after loss of blood flow {Vedder et al., PNAS 87, 2643–2646(1990)}. TNFα also alters the properties of endothelial cells and hasvarious pro-coagulant activities, such as producing an increase intissue factor pro-coagulant activity and suppression of theanticoagulant protein C pathway as well as down-regulating theexpression of thrombomodulin {Sherry et al., J. Cell Biol. 107,1269–1277 (1988)}. TNFα has pro-inflammatory activities which togetherwith its early production (during the initial stage of an inflammatoryevent) make it a likely mediator of tissue injury in several importantdisorders including but not limited to, myocardial infarction, strokeand circulatory shock. Of specific importance may be TNFα-inducedexpression of adhesion molecules, such as intercellular adhesionmolecule (ICAM) or endothelial leukocyte adhesion molecule (ELAM) onendothelial cells {Munro et al., Am. J Path. 135(I), 121–132 (1989)}.

TNFα blockage with monoclonal anti-TNFα antibodies has been shown to bebeneficial in rheumatoid arthritis {Elliot et al., Int. J. Pharmac. 199517(2), 141–145} and Crohn's disease {von Dullemen et al.,Gastroenterology, 1995 109(I), 129–135}

Moreover, it now is known that TNFα is a potent activator of retrovirusreplication including activation of HIV-1. {Duh et al., Proc. Nat. Acad.Sci. 86, 5974–5978 (1989); Poll et al., Proc. Nat. Acad. Sci. 87,782–785 (1990); Monto et al., Blood 79, 2670 (1990); Clouse et al., J.Immunol. 142, 431–438 (1989); Poll et al., AIDS Res. Hum. Retrovirus,191–197 (1992)}. AIDS results from the infection of T lymphocytes withHuman Immunodeficiency Virus (HIV). At least three types or strains ofHIV have been identified; i.e., HIV-1, HIV-2 and HIV-3. As a consequenceof HIV infection, T-cell mediated immunity is impaired and infectedindividuals manifest severe opportunistic infections and/or unusualneoplasms. HIV entry into the T lymphocyte requires T lymphocyteactivation. Other viruses, such as HIV-1, HIV-2 infect T lymphocytesafter T cell activation and such virus protein expression and/orreplication is mediated or maintained by such T cell activation. Once anactivated T lymphocyte is infected with HIV, the T lymphocyte mustcontinue to be maintained in an activated state to permit HIV geneexpression and/or HIV replication. Cytokines, specifically TNFα, areimplicated in activated T-cell mediated HIV protein expression and/orvirus replication by playing a role in maintaining T lymphocyteactivation. Therefore, interference with cytokine activity such as byprevention or inhibition of cytokine production, notably TNFα, in anHIV-infected individual assists in limiting the maintenance of Tlymphocyte caused by HIV infection.

Monocytes, macrophages, and related cells, such as kupffer and glialcells, also have been implicated in maintenance of the HIV infection.These cells, like T cells, are targets for viral replication and thelevel of viral replication is dependent upon the activation state of thecells. {Rosenberg et al., The Immunopathogenesis of HIV Infection,Advances in Immunology, 57 (1989)}. Cytokines, such as TNFα, have beenshown to activate HIV replication in monocytes and/or macrophages {Poliet al., Proc. Natl. Acad. Sci., 87, 782–784 (1990)}; therefore,prevention or inhibition of cytokine production or activity aids inlimiting HIV progression for T cells. Additional studies have identifiedTNFα as a common factor in the activation of HIV in vitro and hasprovided a clear mechanism of action via a nuclear regulator proteinfound in the cytoplasm of cells (Osbom, et al., PNAS 86 2336–2340). Thisevidence suggests that a reduction of TNFα synthesis may have anantiviral effect in HIV infections, by reducing the transcription andthus virus production.

AIDS viral replication of latent HIV in T cell and macrophage lines canbe induced by TNFα {Folks et al., PNAS 86, 2365–2368 (1989)}. Amolecular mechanism for the virus inducing activity is suggested byTNFα's ability to activate a gene regulatory protein (NFκB) found in thecytoplasm of cells, which promotes HIV replication through binding to aviral regulatory gene sequence (LTR) {Osborn et al., PNAS 86, 2336–2340(1989)}. TNFα in AIDS associated cachexia is suggested by elevated serumTNFα and high levels of spontaneous TNFα production in peripheral bloodmonocytes from patients {Wright et al., J. Immunol. 141(I), 99–104(1988)}. TNFα has been implicated in various roles with other viralinfections, such as the cytomegalia virus (CMV), influenza virus,adenovirus, and the herpes family of viruses for similar reasons asthose noted.

The nuclear factor κB (NFκB) is a pleiotropic transcriptional activator(Lenardo, et al., Cell 1989, 58, 227–29). NFκB has been implicated as atranscriptional activator in a variety of disease and inflammatorystates and is thought to regulate cytokine levels including but notlimited to TNFα and also to be an activator of HIV transcription(Dbaibo, et al., J Biol. Chem. 1993, 17762–66; Duh et al., Proc. Natl.Acad. Sci. 1989, 86, 5974–78; Bachelerie et al., Nature 1991, 350,709–12; Boswas et al., J Acquired Immune Deficiency Syndrome 1993, 6,778–786; Suzuki et al., Biochem. And Biophys. Res. Comm. 1993, 193,277–83; Suzuki et al., Biochem. And Biophys. Res. Comm. 1992, 189,1709–15; Suzuki et al., Biochem. Mol. Bio. Int. 1993, 31(4), 693–700;Shakhov et al., Proc. Natl. Acad. Sci. USA 1990, 171, 35–47; and Staalet al., Proc. Natl. Acad. Sci. USA 1990, 87, 9943–47). Thus, inhibitionof NFκB binding can regulate transcription of cytokine gene(s) andthrough this modulation and other mechanisms be useful in the inhibitionof a multitude of disease states. The compounds described herein caninhibit the action of NFκB in the nucleus and thus are useful in thetreatment of a variety of diseases including but not limited torheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, otherarthritic conditions, cancer, septic shock, sepsis, endotoxic shock,graft versus host disease, wasting, Crohn's disease, inflammatory boweldisease, ulcerative colitis, multiple sclerosis, systemic lupuserythrematosis, ENL in leprosy, HIV, AIDS, and opportunistic infectionsin AIDS. TNFα and NFκB levels are influenced by a reciprocal feedbackloop. As noted above, the compounds of the present invention affect thelevels of both TNFα and NFκB.

Decreasing TNFα levels thus constitute valuable therapeutic strategiesfor the treatment of many inflammatory, infectious, immunological ormalignant diseases. These include but are not restricted to septicshock, sepsis, endotoxic shock, hemodynamic shock and sepsis syndrome,post ischemic reperfusion injury, malaria, mycobacterial infection,meningitis, psoriasis, congestive heart failure, fibrotic disease,cachexia, graft rejection, cancer, autoimmune disease, opportunisticinfections in AIDS, rheumatoid arthritis, rheumatoid spondylitis,osteoarthritis, other arthritic conditions, Crohn's disease, ulcerativecolitis, multiple sclerosis, systemic lupus erythrematosis, ENL inleprosy, radiation damage, and hyperoxic alveolar injury.

DETAILED DESCRIPTION

The present invention pertains to compounds of Formula I in which thecarbon atoms designated * constitute centers of chirality:

In Formula I,

-   X is —C(O)— or —CH₂—;-   R¹ is alkyl of 1 to 8 carbon atoms or —NHR³;-   R² is hydrogen, alkyl of 1 to 8 carbon atoms, or halogeno; and-   R³ is hydrogen,    -   alkyl of 1 to 8 carbon atoms,    -   cycloalkyl of 3 to 18 carbon atoms,    -   phenyl, unsubstituted or substituted with halo, amino, or        alkylamino of 1 to 4 carbon atoms,    -   benzyl, unsubstituted or substituted with halo, amino, or        alkylamino of 1 to 4 carbon atoms, or    -   —COR⁴ in which        -   R⁴ is hydrogen,            -   alkyl of 1 to 8 carbon atoms, unsubstituted or                substituted with halo, amino, or alkylamino of 1 to 4                carbon atoms,            -   cycloalkyl of 3 to 18 carbon atoms,            -   phenyl, unsubstituted or substituted with halo, amino,                or alkylamino of 1 to 4 carbon atoms, or            -   benzyl, unsubstituted or substituted with halo, amino,                or alkylamino of 1 to 4 carbon atoms.

The present invention also pertains to the acid addition salts of theseisoindoline derivatives which are susceptible of protonation. Such saltsinclude those derived from organic and inorganic acids such as, withoutlimitation, hydrochloric acid, hydrobromic acid, phosphoric acid,sulfuric acid, methanesulfonic acid, acetic acid, tartaric acid, lacticacid, succinic acid, citric acid, malic acid, maleic acid, sorbic acid,aconitic acid, salicylic acid, phthalic acid, embonic acid, enanthicacid, and the like.

The compounds can be prepared through a number of methods. For example,a suitably protected 3,5-disubstituted piperidine-2,6-dione of FormulaII is allowed to react with a 4-substituted1,3-dihydroisobenzofuran-1,3-dione of Formula III to yield the protectedcompounds of Formula IA:

In the foregoing reaction, R¹ is as defined above, X is —CH₂—, R^(2′) ishydrogen or alkyl, and Z and Y are protecting groups, as for examplebenzyloxycarbonyl and alkanoyloxy.

When X is —CH₂—, a piperidine-2,6-dione of Formula II is allowed toreact with disubstituted alkyl benzoate of Formula IIIA:

Compounds of Formulas III and IIIA are known. Compounds of Formula II inwhich R^(2′) is hydrogen can be prepared by treating an amino protectedlactone of 2-amino-4-hydroxyglutaric acid of Formula IIA with ammonia inmethanol to yield the corresponding protected2-amino-4-hydroxy-4-carboxybutanamide of Formula IIB which is thensubjected to cyclization in acetic acid:

When R^(2′) is alkyl, it can be introduced by treating the lactone ofFormula IIA with two equivalents of a strong base, as for examplen-butyl lithium, to form the dianion, and then alkylating, as forexample with methyl iodide. Alternatively, the unprotected lactone IICis converted to the t.-butyl ester which in turn is treated withbenzaldehyde to form the amidine IID. Treatment of the amidine with baseand an alkyl halide results in alkylation of the α-carbon atom incompound IIE and subsequent treatment with acid cleaves both thet.-butyl ester and amidine yielding the intermediate IIF which can thenbe reprotected as the benzyloxycarbonyl derivative.

When R² is halogeno, as for example fluoro, it can be introduced bytreating a compound of Formula IA or IB with sodiumbis(trimethylsilyl)amide and N-fluorobenzenesulfonimide:

Removal of the protecting group Y can be achieved through appropriatehydrolysis; e.g., treatment with p-toluenesulfonic acid to cleave analkanoyloxy group.

As is apparent from the foregoing, it often is advantageous to utilizedprotected groups including but not limited to functional groupsconvertible to the desired group. Protecting groups utilized hereindenote groups which generally are not found in the final therapeuticcompounds but which are intentionally introduced at some stage of thesynthesis in order to protect groups which otherwise might be altered inthe course of chemical manipulations. Such protecting groups are removedor converted to the desired group at a later stage of the synthesis andcompounds bearing such protecting groups thus are of importanceprimarily as chemical intermediates (although some derivatives alsoexhibit biological activity). Accordingly the precise structure of theprotecting group is not critical. Numerous reactions for the formationand removal of such protecting groups are described in a number ofstandard works including, for example, “Protective Groups in OrganicChemistry”, Plenum Press, London and New York, 1973; Greene, Th. W.“Protective Groups in Organic Synthesis”, Wiley, N.Y., 1981; “ThePeptides”, Vol. I, Schröder and Lubke, Academic Press, London and NewYork, 1965; “Methoden der organischen Chemie”, Houben-Weyl, 4th Edition,Vol. 15/I, Georg Thieme Verlag, Stuttgart 1974, the disclosures of whichare incorporated herein by reference.

An amino group thus can be protected as an amide utilizing an acyl groupwhich is selectively removable under mild conditions, especially formyl,a lower alkanoyl group which is branched in 1- or α position to thecarbonyl group, particularly tertiary alkanoyl such as pivaloyl, or alower alkanoyl group which is substituted in the position α to thecarbonyl group, as for example trifluoroacetyl.

Should a carboxy group require protection, it can be converted to anester which is selectively removable under sufficiently mild conditionsnot to disrupt the desired structure of the molecule, especially a loweralkyl ester of 1 to 12 carbon atoms such as methyl or ethyl andparticularly one which is branched at the 1- or α position such ast-butyl; and such lower alkyl ester substituted in the 1- or 2-positionwith (i) lower alkoxy, such as for example, methoxymethyl,1-methoxyethyl, and ethoxymethyl, (ii) lower alkylthio, such as forexample methylthiomethyl and 1-ethylthioethyl; (iii) halogen, such as2,2,2-trichloroethyl, 2-bromoethyl, and 2-iodoethoxycarbonyl; (iv) oneor two phenyl groups each of which can be unsubstituted or mono-, di- ortri-substituted with, for example lower alkyl such as tert.-butyl, loweralkoxy such as methoxy, hydroxy, halo such as chloro, and nitro, such asfor example, benzyl, 4-nitrobenzyl, diphenylmethyl,di-(4-methoxyphenyl)methyl; or (v) aroyl, such as phenacyl. A carboxygroup also can be protected in the form of an organic silyl group suchas trimethylsilylethyl or tri-lower alkylsilyl, as for exampletri-methylsilyloxycarbonyl.

When R¹ is amino, the reactions described herein can be performed withintermediates in which R¹ is a nitro group with the nitro group thenbeing catalytically reduced (hydrogenated) to an amine. Similarly whenR¹ is derivative of an amino group, such as N-acylamino or N-alkylaminoit can be formed from the corresponding unsubstituted amino compound.

The compounds contain two centers of chirality (designated by * inFormula I) and thus can exist as enantiomers and diastereoisomers. Thecompounds can be administered as a substantially chirally pure (S,S)-,(S,R)-, (R,R)-, or (R,S)-isomer or as mixtures of two or more of theseisomers and all are within the scope of the present invention. Mixturescan be used as such or can be separated into their individual isomersmechanically as by chromatography using a chiral absorbent.Alternatively, the individual isomers can be prepared in chiral form orseparated chemically from a mixture by forming salts with a chiral acid,or have such as, the individual enantiomers of 10-camphorsulfonic acid,camphoric acid, bromocamphoric acid, methoxyacetic acid, tartaric acid,diacetyltartaric acid, malic acid, pyrrolidone-5-carboxylic acid, andthe like, and then freeing one or both of the resolved bases, optionallyrepeating the process, so as obtain either or both substantially free ofthe other; i.e., in a form having an optical purity of >95%.

A first preferred subgroup are those compounds of Formula I in which R²is hydrogen, methyl, or fluoro, particularly hydrogen.

A second preferred subgroup are those compounds of Formula I in which R¹is amino.

A third preferred subgroup are those compounds of Formula I in which R¹is methyl.

A fourth preferred subgroup are those compounds of Formula I in which Xis —C(O)—.

A fifth preferred subgroup are those compounds of Formula I in which Xis —CH₂—

A further preferred subgroup are those compounds of Formula I in whichR² is hydrogen, methyl, or fluoro, particularly hydrogen, R¹ is methyl,amino, alkylamino, or acylamino, and X is —C(O)— or —CH₂—.

Inhibition of TNFα and NFκB by these compounds can be convenientlyassayed using methods known in the art, e.g., enzyme immunoassay,radioimmunoassay, immunoelectrophoresis, affinity labeling, etc., ofwhich the following are typical.

Representative compounds include2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-(N-benzylamino)isoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-(N-benzylamino)isoindolin-1-one;2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-acetamidoisoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-acetamidoisoindolin-1-one;2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-aminoisoindolin-1-one;2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-aminoisoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-methylaminoisoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-methylaminoisoindolin-1-one;2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-methylisoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-methylisoindolin-1-one;2-(2,6-dioxo-3-hydroxy-5-methylpiperidin-5-yl)-4-(N-benzylamino)isoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxy-5-methylpiperidin-5-yl)-4-(N-benzylamino)isoindolin-1-one;2-(2,6-dioxo-3-hydroxy-5-methylpiperidin-5-yl)-4-acetamidoisoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxy-5-methylpiperidin-5-yl)-4-acetamidoisoindolin-1-one;2-(2,6-dioxo-3-hydroxy-5-methylpiperidin-5-yl)-4-aminoisoindolin-1-one;2-(2,6-dioxo-3-hydroxy-5-methylpiperidin-5-yl)-4-aminoisoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxy-5-methylpiperidin-5-yl)-4-methylaminoisoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxy-5-methylpiperidin-5-yl)-4-methylaminoisoindolin-1-one;2-(2,6-dioxo-3-hydroxy-5-methylpiperidin-5-yl)-4-methylisoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxy-5-methylpiperidin-5-yl)-4-methylisoindolin-1-one;2-(2,6-dioxo-3-hydroxypiperidin-5-yl)-4-(N-benzylamino)isoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxypiperidin-5-yl)-4-(N-benzylamino)isoindolin-1-one;2-(2,6-dioxo-3-hydroxypiperidin-5-yl)-4-acetamidoisoindolin-1-one;2-(2,6-dioxo-3-hydroxypiperidin-5-yl)-4-acetamidoisoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxypiperidin-5-yl)-4-aminoisoindolin-1-one;2-(2,6-dioxo-3-hydroxypiperidin-5-yl)-4-aminoisoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxypiperidin-5-yl)-4-methylaminoisoindolin-1-one;2-(2,6-dioxo-3-hydroxypiperidin-5-yl)-4-methylaminoisoindoline-1,3-dione;2-(2,6-dioxo-3-hydroxypiperidin-5-yl)-4-methylisoindolin-1-one; and2-(2,6-dioxo-3-hydroxypiperidin-5-yl)-4-methylisoindoline-1,3-dione.

PBMC from normal donors are obtained by Ficoll-Hypaque densitycentrifugation. Cells are cultured in RPMI supplemented with 10% AB+serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin.

The test compounds are dissolved in dimethylsulfoxide (Sigma Chemical),further dilutions are done in supplemented RPMI. The finaldimethylsulfoxide concentration in the presence or absence of drug inthe PBMC suspensions is 0.25 wt %. The test compounds are assayed athalf-log dilutions starting at 50 mg/mL. The test compounds are added toPBMC (10⁶ cells/mL) in 96 wells plates one hour before the addition ofLPS.

PBMC (10⁶ cells/mL) in the presence or absence of test compound arestimulated by treatment with 1 mg/mL of LPS from Salmonella minnesotaR595 (List Biological Labs, Campbell, Calif.). Cells are then incubatedat 37° C. for 18–20 hours. Supernatants are harvested and assayedimmediately for TNFα levels or kept frozen at −70° C. (for not more than4 days) until assayed.

The concentration of TNFα in the supernatant is determined by human TNFαELISA kits (ENDOGEN, Boston, Mass.) according to the manufacturer'sdirections.

The compounds can be used, under the supervision of qualifiedprofessionals, to inhibit the undesirable effects of TNFα and NFκB. Thecompounds can be administered orally, rectally, or parenterally, aloneor in combination with other therapeutic agents including antibiotics,steroids, etc., to a mammal in need of treatment. Oral dosage formsinclude tablets, capsules, dragees, and similar shaped, compressedpharmaceutical forms. Isotonic saline solutions containing 20–100milligrams/milliliter can be used for parenteral administration whichincludes intramuscular, intrathecal, intravenous and intra-arterialroutes of administration. Rectal administration can be effected throughthe use of suppositories formulated from conventional carriers such ascocoa butter.

Dosage regimens must be titrated to the particular indication, the age,weight, and general physical condition of the patient, and the responsedesired but generally doses will be from about 1 to about 1000milligrams/day as needed in single or multiple daily administration. Ingeneral, an initial treatment regimen can be copied from that known tobe effective in interfering with TNFα activity for other TNFα mediateddisease states by the compounds of the present invention. Treatedindividuals will be regularly checked for T cell numbers and T4/T8ratios and/or measures of viremia such as levels of reversetranscriptase or viral proteins, and/or for progression ofcytokine-mediated disease associated problems such as cachexia or muscledegeneration. If no effect is observed following the normal treatmentregimen, then the amount of cytokine activity interfering agentadministered is increased, e.g., by fifty percent a week.

The compounds of the present invention can also be used topically in thetreatment or prophylaxis of topical disease states mediated orexacerbated by excessive TNFα production, such as viral infections, forexample those caused by the herpes viruses or viral conjunctivitis,psoriasis, other skin disorders and diseases, etc.

The compounds can also be used in the veterinary treatment of mammalsother than humans in need of prevention or inhibition of TNFαproduction. TNFα mediated diseases for treatment, therapeutically orprophylactically, in animals include disease states such as those notedabove, but in particular viral infections. Examples include felineimmunodeficiency virus, equine infectious anaemia virus, caprinearthritis virus, visna virus, and maedi virus, as well as otherlentiviruses.

The invention thus includes various methods of treatment including themethod of reducing or inhibiting undesirable levels of TNFα, method ofreducing or inhibiting undesirable levels of matrix metalloproteinases,the method of treating undesirable angiogenesis, the method of treatingcancer, the method of treating inflammatory disease, the method oftreating autoimmune disease, the method of treating arthritis, themethod of treating rheumatoid arthritis, the method of treatinginflammatory bowel disease, the method of treating Crohn's disease, themethod of treating aphthous ulcers, the method of treating cachexia, themethod of treating graft versus host disease, the method of treatingasthma, the method of treating adult respiratory distress syndrome, andthe method of treating acquired immune deficiency syndrome, byadministering to a mammalan an effective amount of a substantiallychirally pure (R)- or (S)-isomer of a compound of Formula I or a mixtureof those isomers. While these methods may overlap, they also may differin terms of method of administration, dose level, dosage regimen (suchas single or multiple doses), and concurrently administered therapeuticagents.

The invention also includes pharmaceutical compositions in which (i) aquantity of a substantially chirally pure (R)- or (S)-isomer of acompound of Formula I or a mixture of those isomers, that uponadministration in a single or multiple dose regimen is pharmaceuticallyeffective is combined with (ii) a pharmaceutically acceptable carrier.

Pharmaceutical compositions can be typified by oral dosage forms thatinclude tablets, capsules, dragees, and similar shaped, compressedpharmaceutical forms containing from 1 to 100 mg of drug per unitdosage. Mixtures containing from 20 to 100 mg/mL can be formulated forparenteral administration which includes intramuscular, intrathecal,intravenous and intra-arterial routes of administration. Rectaladministration can be effected through the use of suppositoriesformulated from conventional carriers such as cocoa butter.

Pharmaceutical compositions will comprise one or more compounds of thepres??ent invention associated with at least one pharmaceuticallyacceptable carrier, diluent or excipient. In preparing suchcompositions, the active ingredients are usually mixed with or dilutedby an excipient or enclosed within such a carrier which can be in theform of a capsule or sachet. When the excipient serves as a diluent, itmay be a solid, semi-solid, or liquid material which acts as a vehicle,carrier, or medium for the active ingredient. Thus, the compositions canbe in the form of tablets, pills, powders, elixirs, suspensions,emulsions, solutions, syrups, soft and hard gelatin capsules,suppositories, sterile injectable solutions and sterile packagedpowders. Examples of suitable excipients include lactose, dextrose,sucrose, sorbitol, mannitol, starch, gum acacia, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidinone polyvinylpyrrolidone,cellulose, water, syrup, and methyl cellulose, the formulations canadditionally include lubricating agents such as talc, magnesium stearateand mineral oil, wetting agents, emulsifying and suspending agents,preserving agents such as methyl- and propylhydroxybenzoates, sweeteningagents or flavoring agents.

The compositions preferably are formulated in unit dosage form, meaningphysically discrete units suitable as a unitary dosage, or apredetermined fraction of a unitary dose to be administered in a singleor multiple dosage regimen to human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with a suitablepharmaceutical excipient. The compositions can be formulated so as toprovide an immediate, sustained or delayed release of active ingredientafter administration to the patient by employing procedures well knownin the art.

The following examples will serve to further typify the nature of thisinvention but should not be construed as a limitation in the scopethereof, which scope is defined solely by the appended claims.

EXAMPLE 12-(5-Hydroxy-2,6-dioxopiperid-3-yl))-4-methylisoindoline-1,3-dione

A. 3-(4-Methyl-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine

A mixture of 2,6-dioxo-3-benzyloxycarbonylamino-5-acetoxypiperidine (9g, 28.2 mmol) (U. Teubert et al, Arch. Pharm. Pharm. Med. Chem. (1998)7–12) and Pd/C (10%, 0.9 g) in acetic acid (90 mL) is shaken underhydrogen (50–60 psi) for 3 hours. The suspension is filtered through apad of Celite and washed with acetic acid. To the filtrate is added3-methylphthalic anhydride (4.56 g, 28.2 mmol) and this mixture isheated at reflux for 18 hours. The solvent is removed in vacuo to give3-(4-methyl-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidinewhich can be further purified by column chromatography.

B. 2-(5-Hydroxy-2,6-dioxopiperid-3-yl)-4-methylisoindoline-1,3-dione

A solution of3-(4-methyl-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine (1g, 3.5 mmol) and p-toluenesulfonic acid (0.33 g, 1.8 mmol) in methanol(10 mL) is heated at reflux for 5 hours. The solvent is removed in vacuoto give2-(5-hydroxy-2,6-dioxopiperid-3-yl)-4-methylisoindoline-1,3-dione. Theproduct can be further purified by column chromatography.

EXAMPLE 24-Amino-2-(5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1,3-dione

A. 5-(4-Nitro-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine

A solution of 2,6-dioxo-3-benzyloxycarbonylamino-5-acetoxypiperidine (9g, 28.2 mmol) and Pd/C (10%, 0.9 g) in acetic acid (90 mL) is shakenunder hydrogen (50–60 psi) for 3 hours. The suspension is filteredthrough a pad of Celite and washed with acetic acid. To the filtrate isadded 3-nitrophthalic anhydride (5.4 g, 28.2 mmol), this mixture isheated at reflux for 18 hours, and the solvent is then removed in vacuoto give3-(4-nitro-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine whichcan be further purified by column chromatography.

B. 3-(4-Amino-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine

A solution of3-(4-nitro-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine (1 g,3.1 mmol) and Pd/C (10%, 0.1 g) in methanol (100 mL) is shaken underhydrogen (50–60 psi) for 3 hours. The suspension is filtered through apad of Celite and washed with methanol to give3-(4-amino-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine whichcan be further purified by column chromatography.

C. 4-Amino-2-(5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1,3-dione

A solution of3-(4-amino-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine (1 g,3.5 mmol) and p-toluenesulfonic acid (0.33 g, 1.8 mmol) in methanol (10mL) is heated at reflux for 5 hours and the solvent is then removed invacuo to give4-amino-2-(5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1,3-dione whichcan be further purified by column chromatography.

EXAMPLE 3 4-Nitro-2-(5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1-one

A. 3-(4-Nitro-1-oxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine

A solution of 2,6-dioxo-3-benzyloxycarbonylamino-5-acetoxypiperidine (9g, 28.2 mmol) and Pd/C (10%, 0.9 g) in acetic acid (90 mL) is shakenunder hydrogen (50–60 psi) for 3 hours. The suspension is filteredthrough a pad of Celite and washed with acetic acid and the solvent isthen removed in vacuo. The residue, triethyl-amine (2.9 g, 28 mmol), andmethyl 2-bromomethyl-3-nitrobenzoate (7.7 g. 28.2 mmol) indimethylformamide (100 mL) is heated at 80° C. for 18 hours. The solventis removed in vacuo to give3-(4-nitro-1-oxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine which canbe further purified by column chromatography.

B. 4-Nitro-2-(5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1-one

A solution of3-(4-nitro-1-oxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine (0.96 g,3.5 mmol) and p-toluenesulfonic acid (0.33 g, 1.8 mmol) in methanol (10mL) is heated at reflux for 5 hours. The solvent is removed in vacuo togive 4-nitro-2-(5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1-one whichis further purified by column chromatography.

EXAMPLE 4 4-Amino-2-(5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1-one

A. 3-(4-Amino-1-oxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine

A solution of3-(4-nitro-1-oxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine (0.9 g,3.1 mmol) and Pd/C (10%, 0.1 g) in methanol (100 mL) is shaken underhydrogen (50–60 psi) for 3 hours, The suspension is filtered through apad of Celite and washed with methanol to give3-(4-amino-1-oxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine which isfurther purified by column chromatography.

B. 4-Amino-2-(5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1-one

A solution of3-(4-amino-1-oxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine (0.96 g,3.5 mmol) and p-toluenesulfonic acid (0.33 g, 1.8 mmol) in methanol (10mL) is heated at reflux for 5 hours. The solvent is removed in vacuo togive 4-amino-2-(5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1-one whichis further purified by column chromatography.

EXAMPLE 53-[1,3-Dioxo-4-benzamidoisoindolin-2-yl]-2,6-dioxo-5-hydroxypiperidine

A.3-[1,3-Dioxo-4-benzamidoisoindolin-2-yl]-2,6-dioxo-5-acetoxypiperidine

A stirred solution of3-(4-amino-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine (1 g,3.5 mmol) and benzoyl chloride (0.5 g, 3.5 mmol) in tetrahydrofuran (15mL) is heated at reflux for 1 hour. The solvent is removed in vacuo togive3-[1,3-dioxo-4-benzoyllaminoisoindolin-2-yl]-2,6-dioxo-5-acetoxypiperidinewhich is further purified by column chromatography.

B.3-[1,3-Dioxo-4-benzamidoisoindolin-2-yl]-2,6-dioxo-5-hydroxypiperidine

A solution of3-[1,3-dioxo-4-benzamidoisoindolin-2-yl]-2,6-dioxo-5-acetoxypiperidine(1.36 g, 3.5 mmol) and p-toluenesulfonic acid (0.33 g, 1.8 mmol) inmethanol (20 mL) is heated at reflux for 5 hours. The solvent is removedin vacuo to give3-[1,3-dioxo-4-benzamidoisoindolin-2-yl]-2,6-dioxo-5-hydroxypiperidinewhich is further purified by column chromatography.

EXAMPLE 63-[4-(2-Furylcarbonylamino)-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-hydroxypiperidine

A.3-[4-(2-Furylcarbonylamino)-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-acetoxypiperidine

A solution of3-(4-amino-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine (1 g,3.5 mmol) and 2-furonyl chloride (0.46 g, 3.5 mmol) in tetrahydrofuran(20 mL) is heated at reflux for 1 hour. The solvent is removed in vacuoto give3-[4-(2-furyl-carbonylamino)-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-acetoxypiperidinewhich is further purified by column chromatography.

B.3-[4-(2-Furylcarbonylamino)-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-hydroxypiperidine

A solution of3-[4-(2-furylcarbonylamino)-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-acetoxypiperidine(1.33 g, 3.5 mmol) and p-toluenesulfonic acid (0.33 g, 1.8 mmol) inmethanol (20 mL) is heated at reflux for 5 hours. The solvent is removedin vacuo to give3-[4-(2-furylcarbonylamino)-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-hydroxypiperidinewhich is further purified by column chromatography.

EXAMPLE 73-[4-Methoxyacetylamino-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-hydroxypiperidine

A.3-[4-Methoxyacetylamino-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-acetoxypiperidine

A solution of3-(4-amino-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine (1 g,3.5 mmol) and methoxyacetyl chloride (0.38 g, 3.5 mmol) intetrahydrofuran (20 mL) is heated at reflux for 1 hour. The solvent isremoved in vacuo to give3-[4-(2-methoxyacetylamino)-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-acetoxypiperidinewhich is further purified by column chromatography.

B.3-[4-Methoxyacetylamino-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-hydroxypiperidine

A solution of3-[4-methoxyacetylamino-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-acetoxypiperidine(1.26 g, 3.5 mmol) and p-toluenesulfonic acid (0.33 g, 1.8 mmol) inmethanol (20 mL) is heated at reflux for 5 hours. The solvent is removedin vacuo to give3-[4-methoxyacetylamino-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-hydroxypiperidinewhich is further purified by column chromatography.

EXAMPLE 83-(4-Fur-2-ylmethylamino-1,3-dioxoisoindolin-2-yl)-5-hydroxypiperidine-2,6-dione

A solution of3-(4-amino-1,3-dioxoisoindolin-2-yl)-5-hydroxypiperidine-2,6-dione (0.82g, 3.0 mmol) and 2-furaldehyde (0.34 g, 3.5 mmol) in acetic acid (10 mL)is heated at reflux for 4 hours. To the mixture is added sodiumborohydride (130 mg, 3.5 mmol) at room temperature and this mixture ismaintained for 18 hours. The mixture is worked up to give3-(4-fur-2-ylmethylamino-1,3-dioxoisoindolin-2-yl)-5-hydroxypiperidine-2,6-dionewhich is further purified by column chromatography.

EXAMPLE 93-(4-Fur-2-ylmethylamino-1-oxoisoindolin-2-yl)-5-hydroxypiperidine-2,6-dione

A solution of3-(4-amino-1-oxoisoindolin-2-yl)-5-hydroxypiperidine-2,6-dione (0.82 g,3.0 mmol) and 2-furaldehyde (0.34 g, 3.5 mmol) in acetic acid (10 mL) isheated at reflux for 4 hours. To the mixture is added sodium borohydride(130 mg, 3.5 mmol) at room temperature and kept for 18 hours. Themixture is worked up to give3-(4-fur-2-ylmethylamino-1-oxoisoindolin-2-yl)-5-hydroxypiperidine-2,6-dionewhich is further purified by column chromatography.

EXAMPLE 104-Nitro-2-(3-fluoro-5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1,3-dione

A.1-Tert-butoxycarbonyl-3-(4-nitro-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine

To a stirred suspension of3-(4-nitro-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine (2.5g, 7.75 mmol) and di-tert-butyl dicarbonate (1.86 g, 8.52 mmol) in1,4-dioxane (30 mL) is added DMAP (100 mg) at room temperature. Thesolution is stirred at room temperature for 18 hours. The solvent isremoved in vacuo to give1-tert-butoxycarbonyl-3-(4-nitro-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidinewhich is further purified by column chromatography or recrystallization.

A.3-fluoro-3-(4-nitro-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine

To a stirred solution of1-tert-butoxycarbonyl]-3-(4-nitro-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine(2.0 g, 4.3 mmol) in tetrahydrofuran (20 mL) is added sodiumbis(trimethylsilyl)amide (4.3 mL, 4.3 mmol, 1.0 M) in tetrahydrofuran at−78° C. After 10–30 minutes, N-fluorobenzenesulfonimide (1.1 g, 4.3mmol) is added to the mixture. The mixture is warmed to room temperatureand the solvent is removed in vacuo. The residue is stirred with ethylacetate (10 mL) and hydrochloric acid (10 mL, 1N) for 1 hour, theorganic layer is separated, and the solvent is removed in vacuo to give3-fluoro-3-(4-nitro-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidinewhich is further purified by column chromatography.

B.4-Nitro-2-(3-fluoro-5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1,3-dione

A solution of3-(4-nitro-1,3-dioxoisoindolin-2-yl)-3-fluoro-2,6-dioxo-5-acetoxypiperidine(1 g, 2.9 mmol) and p-toluenesulfonic acid (0.28 g, 1.5 mmol) inmethanol (10 mL) is heated at reflux for 5 h. The solvent is removed invacuo to give4-nitro-2-(3-fluoro-5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1,3-dionewhich is further purified by column chromatography.

EXAMPLE 114-Amino-2-(3-fluoro-5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1,3-dione

A.3-(4-Amino-1,3-dioxoisoindolin-2-yl)-3-fluoro-2,6-dioxo-5-acetoxypiperidine

A solution of3-fluoro-3-(4-nitro-1,3-dioxoisoindolin-2-yl)-2,6-dioxo-5-acetoxypiperidine(1.0 g, 2.6 mmol) and Pd/C (10%, 0.1 g) in methanol (100 mL) is shakenunder hydrogen (50–60 psi) for 3 hours. The suspension is filteredthrough a pad of Celite and washed with methanol to give3-(4-amino-1,3-dioxoisoindolin-2-yl)-3-fluoro-2,6-dioxo-5-acetoxypiperidinewhich is further purified by column chromatography.

B.4-Amino-2-(3-fluoro-5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1,3-dione

A solution of3-(4-amino-1,3-dioxoisoindolin-2-yl)-3-fluoro-2,6-dioxo-5-acetoxypiperidine(1 g, 2.9 mmol) and p-toluenesulfonic acid. (0.28 g, 1.5 mmol) inmethanol (10 mL) is heated at reflux for 5 h. The solvent is removed invacuo to give4-amino-2-(3-fluoro-5-hydroxy-2,6-dioxopiperid-3-yl)isoindoline-1,3-dionewhich is further purified by column chromatography.

EXAMPLE 12

Tablets, each containing 50 mg of2-(2,6-dioxo-3-hydroxypiperidin-5-yl)-4-aminoisoindoline-1,3-dione, canbe prepared in the following manner:

Constituents (for 1000 tablets) 2-(2,6-dioxo-3-hydroxypiperidin- 50.0 g5-yl)-4-aminoisoindoline- 1,3-dione lactose 50.7 g wheat starch  7.5 gpolyethylene glycol 6000  5.0 g talc  5.0 g magnesium stearate  1.8 gdemineralized water q.s.

The solid ingredients are first forced through a sieve of 0.6 mm meshwidth. The active ingredient, lactose, talc, magnesium stearate and halfof the starch then are mixed. The other half of the starch is suspendedin 40 mL of water and this suspension is added to a boiling solution ofthe polyethylene glycol in 100 mL of water. The resulting paste is addedto the pulverulent substances and the mixture is granulated, ifnecessary with the addition of water. The granulate is dried overnightat 35° C., forced through a sieve of 1.2 mm mesh width and compressed toform tablets of approximately 6 mm diameter which are concave on bothsides.

EXAMPLE 13

Tablets, each containing 100 mg of2-(2,6-dioxo-3-hydroxypiperidin-5-yl)-4-methylaminoisoindolin-1-one, canbe prepared in the following manner:

Constituents (for 1000 tablets) 2-(2,6-dioxo-3-hydroxypiperidin- 100.0 g5-yl)-4-methylaminoisoindolin- 1-one lactose 100.0 g wheat starch  47.0g magnesium stearate  3.0 g

All the solid ingredients are first forced through a sieve of 0.6 mmmesh width. The active ingredient, lactose, magnesium stearate and halfof the starch then are mixed. The other half of the starch is suspendedin 40 mL of water and this suspension is added to 100 mL of boilingwater. The resulting paste is added to the pulverulent substances andthe mixture is granulated, if necessary with the addition of water. Thegranulate is dried overnight at 35° C., forced through a sieve of 1.2 mmmesh width and compressed to form tablets of approximately 6 mm diameterwhich are concave on both sides.

EXAMPLE 14

Tablets for chewing, each containing 75 mg of2-(2,6-dioxo-3-hydroxy-5-methylpiperidin-5-yl)-4-methylisoindoline-1,3-dione,can be prepared in the following manner:

Composition (for 1000 tablets) 2-(2,6-dioxo-3-hydroxy-  75.0 g5-methylpiperidin-5-yl)- 4-methylisoindoline- 1,3-dione mannitol 230.0 glactose 150.0 g talc  21.0 g glycine  12.5 g stearic acid  10.0 gsaccharin  1.5 g 5% gelatin solution q.s.

All the solid ingredients are first forced through a sieve of 0.25 mmmesh width. The mannitol and the lactose are mixed, granulated with theaddition of gelatin solution, forced through a sieve of 2 mm mesh width,dried at 50° C. and again forced through a sieve of 1.7 mm mesh width.3-(3-Ethoxy-4-methoxyphenyl)-N-hydroxy-3-phthalimidopropionamide, theglycine and the saccharin are carefully mixed, the mannitol, the lactosegranulate, the stearic acid and the talc are added and the whole ismixed thoroughly and compressed to form tablets of approximately 10 mmdiameter which are concave on both sides and have a breaking groove onthe upper side.

EXAMPLE 15

Tablets, each containing 10 mg2-(2,6-dioxo-3-hydroxypiperidin-5-yl)-4-amino-isoindolin-1-one, can beprepared in the following manner:

Composition (for 1000 tablets) 2-(2,6-dioxo-3-hydroxypiperidin-  10.0 g5-yl)-4-aminoisoindolin- 1-one lactose 328.5 g corn starch  17.5 gpolyethylene glycol 6000  5.0 g talc  25.0 g magnesium stearate  4.0 gdemineralized water q.s.

The solid ingredients are first forced through a sieve of 0.6 mm meshwidth. Then the active imide ingredient, lactose, talc, magnesiumstearate and half of the starch are intimately mixed. The other half ofthe starch is suspended in 65 mL of water and this suspension is addedto a boiling solution of the polyethylene glycol in 260 mL of water. Theresulting paste is added to the pulverulent substances, and the whole ismixed and granulated, if necessary with the addition of water. Thegranulate is dried overnight at 35° C., forced through a sieve of 1.2 mmmesh width and compressed to form tablets of approximately 10 mmdiameter which are concave on both sides and have a breaking notch onthe upper side.

EXAMPLE 16

Gelatin dry-filled capsules, each containing 100 mg of2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-aminoisoindolin-1-one,can be prepared in the following manner:

Composition (for 1000 capsules) 2-(2,6-dioxo-3-hydroxy- 100.0 g5-fluoropiperidin-5-yl)- 4-aminoisoindolin-1-one microcrystallinecellulose  30.0 g sodium lauryl sulfate  2.0 g magnesium stearate  8.0 g

The sodium lauryl sulfate is sieved into the2-(2,6-dioxo-3-hydroxy-5-fluoropiperidin-5-yl)-4-aminoisoindolin-1-onethrough a sieve of 0.2 mm mesh width and the two components areintimately mixed for 10 minutes. The microcrystalline cellulose is thenadded through a sieve of 0.9 mm mesh width and the whole is againintimately mixed for 10 minutes. Finally, the magnesium stearate isadded through a sieve of 0.8 mm width and, after mixing for a further 3minutes, the mixture is introduced in portions of 140 mg each into size0 (elongated) gelatin dry-fill capsules.

EXAMPLE 17

A 0.2% injection or infusion solution can be prepared, for example, inthe following manner:

2-(2,6-dioxo-3-hydroxypiperidin-  5.0 g 5-yl)-4-aminoisoindolin- 1-onehydrochloride sodium chloride  22.5 g phosphate buffer pH 7.4 300.0 gdemineralized water to 2500.0 mL

2-(2,6-Dioxo-3-hydroxypiperidin-5-yl)-4-aminoisoindolin-1-onehydrochloride is dissolved in 1000 mL of water and filtered through amicrofilter. The buffer solution is added and the whole is made up to2500 mL with water. To prepare dosage unit forms, portions of 1.0 or 2.5mL each are introduced into glass ampoules (each containing respectively2.0 or 5.0 mg of imide).

1.3-[1,3-Dioxo-4-benzamidoisoindolin-2-yl]-2,6-dioxo-5-hydroxypiperidine.2.3-[4-(2-Furylcarbonylamino)-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-hydroxypiperidine.3.3-[4-Methoxyacetylamino-1,3-dioxoisoindolin-2-yl]-2,6-dioxo-5-hydroxypiperidine.4.3-(4-Fur-2-ylmethylamino-1,3-dioxoisoindolin-2-yl)-5-hydroxypiperidine-2,6-dione.5.3-(4-Fur-2-ylmethylamino-oxoisoindolin-2-yl)-5-hydroxypiperidine-2,6-dione.